Filtered with samtools flag 1804 (samtools view -F 1804):
read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)
Fraction of mitochondrial reads (unfiltered BAM)
rep1
rep2
Rn = Number of Non-mitochondrial Reads
33198628
99465529
Rm = Number of Mitochondrial Reads
184786
618828
Rm/(Rn+Rm) = Frac. of mitochondrial reads
0.005535263709098176
0.006183064152572814
SAMstat (filtered/deduped BAM)
rep1
rep2
Total Reads
22760276
67614240
Total Reads (QC-failed)
0
0
Duplicate Reads
0
0
Duplicate Reads (QC-failed)
0
0
Mapped Reads
22760276
67614240
Mapped Reads (QC-failed)
0
0
% Mapped Reads
100.0
100.0
Paired Reads
22760276
67614240
Paired Reads (QC-failed)
0
0
Read1
11380138
33807120
Read1 (QC-failed)
0
0
Read2
11380138
33807120
Read2 (QC-failed)
0
0
Properly Paired Reads
22760276
67614240
Properly Paired Reads (QC-failed)
0
0
% Properly Paired Reads
100.0
100.0
With itself
22760276
67614240
With itself (QC-failed)
0
0
Singletons
0
0
Singletons (QC-failed)
0
0
% Singleton
0.0
0.0
Diff. Chroms
0
0
Diff. Chroms (QC-failed)
0
0
Filtered and duplicates are removed.
Subsampling with atac.subsample_reads is not done in alignment steps.
Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled
with such parameter in the peak-calling step.
Fragment length statistics (filtered/deduped BAM)
rep1
rep2
Fraction of reads in NFR
0.7115782862880241
0.7100783510400204
Fraction of reads in NFR (QC pass)
True
True
Fraction of reads in NFR (QC reason)
OK
OK
NFR / mono-nuc reads
2.690391927331464
2.6385425585045663
NFR / mono-nuc reads (QC pass)
True
True
NFR / mono-nuc reads (QC reason)
OK
OK
Presence of NFR peak
True
True
Presence of Mono-Nuc peak
True
True
Presence of Di-Nuc peak
False
False
rep1rep2
Open chromatin assays show distinct fragment length enrichments, as the cut
sites are only in open chromatin and not in nucleosomes. As such, peaks
representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal)
fragment lengths will arise. Good libraries will show these peaks in a
fragment length distribution and will show specific peak ratios.
NFR: Nucleosome free region
Sequence quality metrics (filtered/deduped BAM)
rep1rep2
Open chromatin assays are known to have significant GC bias. Please take this
into consideration as necessary.
Annotated genomic region enrichment
rep1
rep2
Fraction of Reads in universal DHS regions
0.5968982977183581
0.578667008606471
Fraction of Reads in blacklist regions
0.006021499915027393
0.003990283703551205
Fraction of Reads in promoter regions
0.1798394711909469
0.17362450572542115
Fraction of Reads in enhancer regions
0.412723202477861
0.4024473394953489
Signal to noise can be assessed by considering whether reads are falling into
known open regions (such as DHS regions) or not. A high fraction of reads
should fall into the universal (across cell type) DHS set. A small fraction
should fall into the blacklist regions. A high set (though not all) should
fall into the promoter regions. A high set (though not all) should fall into
the enhancer regions. The promoter regions should not take up all reads, as
it is known that there is a bias for promoters in open chromatin assays.
Library complexity quality metrics
Library complexity (filtered non-mito BAM)
rep1
rep2
Total Fragments
12023511
36196537
Distinct Fragments
11390159
33867478
Positions with Two Read
491605
1671907
NRF = Distinct/Total
0.947324
0.935655
PBC1 = OneRead/Distinct
0.95317
0.944815
PBC2 = OneRead/TwoRead
22.084322
19.138931
Mitochondrial reads are filtered out by default.
The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads
in a dataset; it is the ratio between the number of positions in the genome
that uniquely mapped reads map to and the total number of uniquely mappable
reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations
with EXACTLY one read pair over the genomic locations with AT LEAST one read
pair. PBC1 is the primary measure, and the PBC1 should be close to 1.
Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking,
0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is
the ratio of genomic locations with EXACTLY one read pair over the genomic
locations with EXACTLY two read pairs. The PBC2 should be significantly
greater than 1.
Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally
N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'
Number of raw peaks
rep1
rep2
Number of peaks
153507
199099
The number of peaks is capped at 300000 Peaks are called from macs2 with p-val threshold 0.01
Peak calling statistics
Peak region size
rep1
rep2
idr_opt
overlap_opt
Min size
150.0
150.0
150.0
150.0
25 percentile
191.0
209.0
486.0
343.0
50 percentile (median)
286.0
332.0
745.0
538.0
75 percentile
520.0
626.0
1094.0
876.0
Max size
2444.0
3082.0
3087.0
3087.0
Mean
408.490765893412
479.91451991220447
824.6770124664436
659.4317484044464
rep1rep2idr_optoverlap_opt
Enrichment / Signal-to-noise ratio
TSS enrichment (filtered/deduped BAM)
rep1
rep2
TSS enrichment
12.868879168579248
12.728467987530006
rep1rep2
Open chromatin assays should show enrichment in open chromatin sites, such as
TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is
above 10. For other references please see https://www.encodeproject.org/atac-seq/
Jensen-Shannon distance (filtered/deduped BAM)
rep1
rep2
AUC
0.12161090374617473
0.16077010805100822
Synthetic AUC
0.48996389787314965
0.49418751744417083
X-intercept
0.39046278374098187
0.17221617903476077
Synthetic X-intercept
2.758692794537362e-84
6.740138456396787e-254
Elbow Point
0.7484923135987714
0.7641490569961448
Synthetic Elbow Point
0.49922269426240773
0.5055456531927898
Synthetic JS Distance
0.456881003096886
0.4749334025884419
Peak enrichment
Fraction of reads in peaks (FRiP)
FRiP for macs2 raw peaks
rep1
rep2
rep1-pr1
rep2-pr1
rep1-pr2
rep2-pr2
pooled
pooled-pr1
pooled-pr2
Fraction of Reads in Peaks
0.3235066657363909
0.3221814073485112
0.31360674185146087
0.30747324823883254
0.312779335364826
0.30804851167446384
0.3337652065544672
0.315363083106304
0.3151767473919307
FRiP for overlap peaks
rep1_vs_rep2
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks
0.29460196500526764
0.27287393175724234
0.2886000049693674
0.3018841063558227
FRiP for IDR peaks
rep1_vs_rep2
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks
0.24649442106002536
0.19988544954375773
0.2413224344457617
0.25640433858589073
For macs2 raw peaks:
repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)
For overlap/IDR peaks:
repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates